The present invention relates to methods of administration of recombinant gene transfer vehicles for the treatment of hemophilia, thrombosis, and other diseases. The present invention also relates generally to recombinant retroviruses, and more specifically, to high titer recombinant retroviral particle preparations suitable for a variety of applications.
A variety of human disorders can be treated by the methods described herein. For example, hemophilia is a genetic disease characterized by a severe blood clotting deficiency. As such, it will be amenable to treatment by gene therapy. In hemophilia A, an X-chromosome linked genetic defect disrupts the gene encoding factor VIII, a trace plasma glycoprotein which acts as a cofactor in conjunction with factor IXa in the activation of factor X. In humans, the factor VIII gene codes for 2,351 amino acids. The protein has six domains, designated from amino to carboxy terminus as A1, A2, B, A3, C1, and C2, respectively (Wood et al., 1984, Nature 312:330; Vehar et al., 1984, Nature 312:337; and Toole et al., 1984, Nature 312:342) with a deduced molecular weight of about 280 kilo Daltons (kD). The 980 amino acid B domain is deleted in the activated procoagulant form of the protein. Additionally, in the native protein two polypeptide chains, the heavy and light chain, flanking the B domain, are bound to a divalent calcium cation.
The genetic defect causing hemophilia A affects about one in every 10,000 males. Due to the resultant clotting deficiency, those afflicted with the disease suffer severe bleeding episodes due to small injuries, internal bleeding, and joint hemorrhage, which leads to arthropathy, the major cause of morbidity in hemophilia. Normal levels of factor VIII average between 50 to 200 ng/ml of blood plasma (Mannucci, P. M. in Practical Laboratory Hematology, ed. Koepke, J. A., Churchill Livingstone, N.Y., pp:347-371, 1990); however, patients suffering from mild to moderate hemophilia A typically have plasma levels well below 2-60 ng/ml, while levels below about 2 ng/mL result in severe hemophilia.
Previously, therapy for hemophilia A involved repeated administration off human factor VIII purified from blood products pooled in lots from over 1000 donors. However, because of the low levels of circulating factor VIII, resulting pharmaceutical products using the natural protein typically were highly impure, with an estimated purity by weight (factor VIII to total protein) of approximately 0.04%. Due to the frequency of administration and inability to remove various human pathogens from such preparations, more than 90% of those suffering from hemophilia A were infected in the 1980s with the human immunodeficiency virus (HIV) as a result of their therapy. Many of these HIV infected patients and other HIV negative hemophiliacs have also been infected by Hepatitis B in the same way. Fortunately, recent advances in genetic engineering have lead to the commercial availability of a recombinant form of the protein free from contamination with human pathogens, except for those potentially derived from tissue culture origin of the proteins, or from human serum albumin used in formulation of the recombinant protein. However, this form of therapy is expensive and chronic, and a large proportion of hemophiliacs continue to rely on plasma-derived products due to expense and or shortages of the recombinant product. In addition, most hemophilia A patients in the Unites States do not presently receive factor VIII maintenance therapy, but instead only receive the polypeptide prior to activities or events which might cause bleeding, such as surgery, or to treat spontaneous bleeding. Interestingly, this is despite evidence showing that hemophilic arthropathy can be prevented by administering from an early age prophylactic amounts of factor VIII, typically 24-40 IU per kilogram bodyweight, three times a week. Such therapy kept factor VIII concentrations from falling below 1% of normal (Nillson, et al., J. Internal Med. 232:23, 1992). For these reasons, a genetic therapy affording continuous, long term therapeutically effective expression levels or amounts of factor VIII, i.e., to decrease the severity of or eliminate the clotting disorder associated with hemophilia A, would be of great benefit.
A condition clinically indistinguishable from Hemophilia A is Hemophilia B, resulting from the deficiency of clotting factor IX. The incidence of this condition is about 5-fold lower than that of hemophilia A, and presents many of the same therapeutic challenges and difficulties. For similar reasons, it would be of great benefit to provide a gene therapy to these patients.
Factor X deficiency results in a rare but serious bleeding disorder affecting 1 in 500,000 known as Stuart disease. Le et al., 1997, Blood 89:1254-9, describes therapeutic levels of functional human factor X in rats after retroviral mediated hepatic gene therapy. As in the case of hemophilia A and B, a genetic therapy affording continuous, long term therapeutically effective expression levels or amounts of factor X, i.e., to decrease the severity of or eliminate the clotting disorder associated with hemophilia B, would be of great benefit.
The present invention also provides for gene therapy delivery of other clotting factors for treatment or prophalaxis of thrombosis. Venous thromboembolism has an annual incidence of 1/1000 in the general population (Dahlback, 1995, Blood 85:607). Precipitating factors can include hemostatic challenges such as surgery, fractures, inflammation, immobilization, pregnancy, oral contraceptive use, trauma, cancer, etc. Thrombosis is often familial, and a number of genetic risk factors have been identified. The clinical condition in which recurrent thrombosis occurs has been dubbed thrombophilia. The natural defenses against thrombosis involve two major systems: serpin inhibitors of thrombin, e.g., antithrombin III, the major pathway by which heparin exerts its clinical antithrombin effect, and the protein C system. Gene therapy for thrombosis disorders is needed and is addressed by the instant invention.
The present invention also provides methods for treatment of diseases such as viral hepatitis. Currently, the only approved treatment for chronic hepatitis B, C and D infections is the use of alpha interferon 2a and 2b. However, for patients with hepatitis B infections only about 35% of patients infected as adults responded to such treatment, and in perinatal infectees only about 10% responded to treatment (Perrillo et al., 1990, New Eng. J. Med. 323:295-301). For hepatitis C infections, despite apparent short term success utilizing such therapy, six months after termination of treatment half of the patients who responded to therapy had relapsed. (Davis et al., New Eng. J. Med. 321:1501-1506). In pilot studies for hepatitis D infections, 25-60% of patients responded to alpha interferon therapy. Sustained responses were rare; 85-90% of patients who responded had relapsed. (di Bisceglie, A. M. D., Viral Hepatitis A to F: An Update, 1994). In addition, a further difficulty with alpha interferon therapy is that the composition frequently has toxic side effects which require reduced dosages for sensitive patients. Thus, improved methods for treatment of viral hepatitis are needed and are addressed by the present invention.
The instant invention also relates to the production and use of high titer recombinant retroviruses. Since the discovery of DNA in the 1940s and continuing through the most recent era of recombinant DNA technology, substantial research has been undertaken in order to realize the possibility that the course of disease may be affected through interaction with the nucleic acids of living organisms. Most recently, a wide variety of methods have been described for altering or affecting genes, including for example, viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses (see Jolly, 1994, Cancer Gene Therapy 1(1):51-64), as well as direct transfer techniques such as lipofection (Felgner et al., 1989, Proc. Natl. Acad. Sci. USA 84:7413-7417), direct DNA injection (Acsadi et al., 1991, Nature 352:815-818), microprojectile bombardment (Williams et al., 1991, PNAS 88:2726-2730), liposomes of several types (see, e.g., Wang et al., 1987, PNAS 84:7851-7855) and administration of nucleic acids alone (PCT Patent Publication No. WO 90/11092).
Of these techniques, recombinant retroviral gene delivery methods have been most extensively utilized, in part due to: (1) the efficient entry of genetic material (the vector genome) into cells; (2) an active, efficient process of entry into the target cell nucleus; (3) relatively high levels of gene expression; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific control of gene expression; (5) a general lack of pre-existing host immunity; and (6) substantial knowledge and clinical experience which has been gained with such vectors.
Briefly, retroviruses are diploid positive-strand RNA viruses that replicate through an integrated DNA intermediate. In particular, upon infection by the RNA virus, the retroviral genome is reverse-transcribed into DNA by a virally encoded reverse transcriptase that is carried as a protein in each retrovirus. The viral DNA is then integrated pseudo-randomly into the host cell genome of the infecting cell, forming a xe2x80x9cprovirusxe2x80x9d which is inherited by daughter cells.
Wild-type retroviral genomes (and their proviral copies) contain three genes (the gag, pol and env genes), which are preceded by a packaging signal ("psgr"), and two long terminal repeat (LTR) sequences which flank both ends. Briefly, the gag gene encodes the internal structural (nucleocapsid) proteins. The pol gene codes for the RNA-dependent DNA polymerase which reverse transcribes the RNA genome, and the env gene encodes the retroviral envelope glycoproteins. The 5xe2x80x2 and 3xe2x80x2 LTRs contain cis-acting elements necessary to promote transcription and polyadenylation of retroviral RNA.
Adjacent to the 5xe2x80x2 LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of retroviral RNA into particles (the "psgr" sequence). Removal of the packaging signal prevents encapsidation (packaging of retroviral RNA into infectious virions) of genomic RNA, although the resulting mutant can still direct synthesis of all proteins encoded in the viral genome.
Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., 1983, Cell 33:153; Cane and Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349; Miller et al., 1990, Human Gene Therapy 1:5-14; U.S. Pat. Nos. 4,405,712; 4,861,719; 4,980,289 and PCT Patent Publication Nos. WO 89/02468; WO 89/05349 and WO 90/02806. Briefly, a foreign gene of interest may be incorporated into the retrovirus in place of the normal retroviral RNA. When the retrovirus injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be integrated into the host""s cellular DNA as if it were the retrovirus itself. Expression of this foreign gene within the host results in expression of the foreign protein by the host cell.
One disadvantage, however, of recombinant retroviruses is that they principally infect only replicating cells, thereby making efficient direct gene transfer difficult. Indeed, some scientists have suggested that other, more efficient methods of gene transfer, such as direct administration of pure plasmid DNA, be utilized (Davis et al., 1993, Human Gene Therapy 4:733-740).
In order to increase the efficacy of recombinant retroviruses, methods which have been suggested for increasing the efficacy of recombinant retroviruses have principally been aimed at inducing the desired target cells to replicate, thereby allowing the retroviruses to infect the cells. Such methods have included, for example chemical treatment with 10% carbon tetrachloride in mineral oil (Kaleko et al., 1991, Human Gene Therapy 2:27-32). Alternatively, others have suggested excising large portions of the liver (e.g., 70% in Rettinger et al., 1994 PNAS 91:1460-1464; 70% in Moscioni et al., 1993, Surgery 113:304-311) in order to stimulate the rapid division of hepatocytes and thereby increase the infection of such cells.
One further disadvantage of recombinant retroviruses, is that serum from primates (e.g., humans) is known to cause inactivation by an antibody independent complement lysis method. In particular retroviruses of avian, murine, feline, and simian origin are all inactivated and lysed by normal human serum. (Welsh et al., 1975, Nature 257:612-614; Welsh et al., 1976, Virology 74:432-440; Banapour et al., 1986, Virology 152:268-271; and Cooper et al., Immunology of the Complement System, Pub., American Press, Inc., pp. 139-162, 1986). The scientific literature has also reported that replication competent murine amphotropic retroviruses injected intravenously into primates are cleared within 15 minutes and that the disappearance is mediated, wholly or in part, by primate complement. (Cornetta et al., 1991, Human Gene Therapy 2:5-14; Cornetta et al., 1990, Human Gene Therapy 1:15-30; and Banapour et al., 1986, Virology 152:268-271)
In order to increase the affect of recombinant retroviruses that are delivered in vivo, the present invention provides recombinant retrovirus compositions which are capable of surviving inactivation in human serum. In addition, the present invention provides high titer recombinant retrovirus compositions which allow delivery of therapeutics or palliatives by routes not previously deemed possible, and without the need to induce replication of cells by chemical treatment or by excision of a target organ such as the liver. The present invention provides these, as well as other related advantages.
This invention provides for preparations of replication defective recombinant retrovirus expressing human factor VIII protein, wherein the recombinant retrovirus is capable of infecting human cells and is resistant to degradation by human complement. The invention also provides for preparations of replication defective recombinant retrovirus expressing human factor VIII protein in which the recombinant retrovirus preparation is resistant to degradation by human complement and is capable of inducing long term systemic expression of human factor VIII when administered intravenously to a human afflicted with hemophilia A. The wherein said long term systemic expression results in a measurable level of recombinant human factor VIII protein being produced in the blood of said human for a period of at least 30 days after the administration of said recombinant retroviral vector preparation and more preferably for at least six months after injection, and yet more preferably for longer periods of time as described herein.
Pharmaceutical compositions and therapeutic methods of the above-described retroviral vectors expressing factor VIII protein are also provided herein as are therapeutic methods for treatment of hemophilia A by intravenous injection of these retroviral vectors. The retroviral vectors of the invention can expresses a B domain-deleted form of factor VIII, which in one embodiment can be the SQN mutation of factor VIII. The retroviral vectors of the invention can have a titer on HT1080 cells of greater than 106, more preferably 107 cfu/ml and more preferably at least 108 cfu/ml, more preferably 109 cfu/ml, more preferably at least 1010 cfu/ml, and most preferably 1011 cfu/ml.
In addition, the present invention provides high titer compositions comprising recombinant retroviruses, as well as methods for utilizing these compositions. Within one aspect of the present invention, methods are provided for obtaining measurable levels of a protein, nucleic acid molecule, or enzymatic product in a bodily fluid or cells of a human, comprising the step of administering to a human a recombinant retroviral preparation having a titer on HT1080 cells of greater than 105 cfu/ml, wherein the recombinant retroviral preparation is capable of directing the expression of a protein, nucleic acid molecule, or enzyme which generates an enzymatic product, such that measurable levels of the protein, nucleic acid molecule, or enzymatic product may be obtained in the bodily fluid or cells of said human. Within certain embodiments, the titer may be greater than 106 cfu/ml, 107 cfu/ml, 108 cfu/ml, 109 cfu/ml, 1010 cfu/ml, or 1011 cfu/ml.
Within other aspects of the invention, methods are provided for obtaining measurable levels of a protein, nucleic acid molecule, or enzymatic product in a bodily fluid or cells of a human, comprising the steps of administering to a human a recombinant retroviral preparation having a titer in human serum and on HT1080 cells equivalent to its"" titer in heat-inactivated serum and on HT1080 cells, wherein the recombinant retroviral preparation is capable of directing the expression of a protein, nucleic acid molecule, or enzyme which generates an enzymatic product, such that measurable levels of the protein, nucleic acid molecule, or enzymatic product may be obtained in the bodily fluid or cells of said human.
As utilized within the context of the present invention, xe2x80x9cmeasurable levelsxe2x80x9d of a protein, nucleic acid molecule, or enzymatic product refers to a statistically significant level of detection over background, utilizing any suitable technique (e.g., antibody-mediated detection of a protein, PCR analysis for the presence of a nucleic acid molecule, or visualization of enzymatic products). Further, as utilized within the context of the present invention, xe2x80x9cequivalentxe2x80x9d titers are deemed to be those which are substantially the same within a given assay, generally, within about three-fold of each other.
Within certain embodiments of the invention, the recombinant retrovirus is administered to a site such as the cerebral spinal fluid, bone marrow, joints, arterial endothelial cells, rectum, buccal/sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces. Within other embodiments, the recombinant retrovirus may be administered intraocularly, intranasally, sublinually, orally, topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracranially, intramuscularly, or subcutaneously.
Within yet other embodiments of the present invention, the protein is a viral antigen obtained from a virus such as influenza virus, respiratory syncytial virus, HPV, HBV, HCV, EBV, HIV, HSV, FeLV, FIV, Hantavirus, HTLV I, HTLV II and CMV. Within other embodiments, the protein is a cytokine such as IL-1, IL-2, IL-3, IL4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, xcex3-IFN, G-CSF and GM-CSF, or a receptor for any of these cytokines.
Within another embodiment, the nucleic acid molecule may be an antisense sequence, a non-coding non-heterologous sense sequence, and a ribozyme sequence. Within yet another aspect, the protein is a toxin.